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Modular method to prepare tetrameric cytokines with improved pharmacokinetics by the dock-and-lock (DNL) technology

a technology of tetramer cytokines and modules, applied in the direction of peptides, drug compositions, immunological disorders, etc., can solve the problems of primarily hindering the prognosis of ifn as a cancer treatmen

Active Publication Date: 2011-03-15
IBC PHARMACEUTICALS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

"The present invention is about methods and compositions for creating stable and defined conjugates of cytokines and antibodies using the Dock-and-Lock (DNL) method. These conjugates can be used for cancer therapy and have been shown to be effective in treating tumors in animal models. The DNL method involves attaching two copies of a cytokine to a dimerization and docking domain (DD) moiety, which spontaneously dimerizes and binds to an anchor domain (AD) moiety. The skilled artisan can also incorporate other types of antibodies or antigen-binding fragments into the cytokine-antibody complexes. The patent text describes various types of target antigens that can be targeted by these conjugates. Overall, the invention provides a way to create stable and effective cytokine-antibody complexes for cancer therapy."

Problems solved by technology

The promise of IFNα as a cancer therapeutic has been hindered primarily due to its short circulating half-life and systemic toxicity.

Method used

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  • Modular method to prepare tetrameric cytokines with improved pharmacokinetics by the dock-and-lock (DNL) technology
  • Modular method to prepare tetrameric cytokines with improved pharmacokinetics by the dock-and-lock (DNL) technology
  • Modular method to prepare tetrameric cytokines with improved pharmacokinetics by the dock-and-lock (DNL) technology

Examples

Experimental program
Comparison scheme
Effect test

example 1

CH3-AD2-IgG Expression Vectors

[0130]The pdHL2 mammalian expression vector has been used for the expression of recombinant IgGs (Qu et al., Methods 2005, 36:84-95). A plasmid shuttle vector was produced to facilitate the conversion of any IgG-pdHL2 vector into a CH3-AD2-IgG-pdHL2 vector. The gene for the Fc (CH2 and CH3 domains) was amplified by PCR using the pdHL2 vector as a template and the following oligonucleotide primers:

[0131]

Fc BglII LeftAGATCTGGCGCACCTGAACTCCTG(SEQ ID NO: 1)Fc Bam- EcoRI RightGAATTCGGATCCTTTACCCGGAGACAGGGAGAG.(SEQ ID NO: 2)

[0132]The amplimer was cloned in the pGemT PCR cloning vector (Promega). The Fc insert fragment was excised from pGemT with Xba I and Bam HI and ligated with AD2-pdHL2 vector that was prepared by digesting h679-Fab-AD2-pdHL2 (Rossi et al., Proc Natl Acad Sci USA 2006, 103:6841-6) with Xba I and Bam HI, to generate the shuttle vector Fc-AD2-pdHL2. To convert IgG-pdHL2 expression vectors to a CH3-AD2-IgG-pdHL2 expression vectors, an 861 bp B...

example 2

Production of CH3-AD2-IgG

[0139]Transfection and Selection of Stable CH3-AD2-IgG Secreting Cell Lines

[0140]All cell lines were grown in Hybridoma SFM (Invitrogen, Carlsbad Calif.). CH3-AD2-IgG-pdHL2 vectors (30 μg) were linearized by digestion with Sal I restriction endonuclease and transfected into Sp2 / 0-Ag14 (2.8×106 cells) by electroporation (450 volts, 25 μF). The pdHL2 vector contains the gene for dihydrofolate reductase allowing clonal selection as well as gene amplification with methotrexate (MTX).

[0141]Following transfection, the cells were plated in 96-well plates and transgenic clones were selected in media containing 0.2 μM MTX. Clones were screened for CH3-AD2-IgG productivity by a sandwich ELISA using 96-well microtitre plates coated with specific anti-idiotype MAbs. Conditioned media from the putative clones were transferred to the micro-plate wells and detection of the fusion protein was accomplished with horseradish peroxidase-conjugated goat anti-human IgG F(ab′)2 (J...

example 3

Generation of DDD-Module based on Interferon (IFN)-α2b

[0144]Construction of IFN-α2b-DDD2-pdHL2 for Expression in Mammalian Cells

[0145]The cDNA sequence for IFN-α2b was amplified by PCR resulting in sequences comprising the following features, in which XbaI and BamHI are restriction sites, the signal peptide is native to IFN-α2b, and 6 His is a hexahistidine tag: XbaI---Signal peptide---IFNα2b---6 His---BamHIH (6 His disclosed as SEQ ID NO: 28). The resulting secreted protein consisted of IFN-α2b fused at its C-terminus to a polypeptide of the following sequence:

[0146]

(SEQ ID NO: 3)KSHHHHHHGSGGGGSGGGCGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA.

[0147]PCR amplification was accomplished using a full length human IFNα2b cDNA clone (Invitrogen Ultimate ORF human clone cat# HORF01 Clone ID IOH35221) as a template and the following oligonucleotides as primers:

[0148]

IFNA2 Xba I Left(SEQ ID NO: 4)TCTAGACACAGGACCTCATCATGGCCTTGACCTTTGCTTTACTGGIFNA2 BamHI right(SEQ ID NO: 5)GGATCCATGATGGTGATGAT...

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Abstract

The present invention concerns methods and compositions for forming cytokine-antibody complexes using dock-and-lock technology. In preferred embodiments, the cytokine-MAb DNL complex comprises an IgG antibody attached to two AD (anchor domain) moieties and four cytokines, each attached to a DDD (docking and dimerization domain) moiety. The DDD moieties form dimers that bind to the AD moieties, resulting in a 2:1 ratio of DDD to AD. The cytokine-MAb complex exhibits improved pharmacokinetics, with a significantly longer serum half-life than either naked cytokine or PEGylated cytokine. The cytokine-MAb complex also exhibits significantly improved in vitro and in vivo efficacy compared to cytokine alone, antibody alone, unconjugated cytokine plus antibody or cytokine-MAb DNL complexes incorporating an irrelevant antibody. In a most preferred embodiment the complex comprises an anti-CD20 IgG antibody conjugated to four IFN-α2b moieties, although other antibodies and cytokines have been used to form effect DNL complexes.

Description

RELATED APPLICATIONS[0001]This application claims the benefit under 35 U.S.C. 119(e) of U.S. Provisional Patent Application Ser. Nos. 61 / 043,932, filed Apr. 10, 2008, 61 / 104,916, filed Oct. 13, 2008 and 61 / 119,542, filed Dec. 3, 2008. This application is a continuation-in-part of U.S. patent application Ser. No. 12 / 396,965, filed Mar. 3, 2009; which was a divisional of U.S. Ser. No. 11 / 391,584 (now U.S. Pat. No. 7,521,056), filed Mar. 28, 2006; U.S. Ser. No. 11 / 389,358 (now U.S. Pat. No. 7,550,143), filed Mar. 24, 2006; U.S. Ser. No. 11 / 478,021 (now U.S. Pat. No. 7,534,866), filed Jun. 29, 2006; U.S. Ser. No. 12 / 396,605, filed Mar. 3, 2009, which was a divisional of Ser. No. 11 / 633,729 (now U.S. Pat. No. 7,527,787), filed Dec. 5, 2006; Ser. No. 11 / 745,692, filed May 8, 2007; and U.S. Ser. No. 11 / 925,408 (now U.S. Pat. No. 7,666,400), filed Oct. 26, 2007. Those applications claimed the benefit under 35 U.S.C. 119(e) of U.S. Provisional Patent Application Ser. Nos. 60 / 668,603, filed A...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): A61K39/395C07K14/00A61K39/44A61K38/19C12P21/08C07K16/46C07K14/52A61K38/20A61K38/21
CPCA61K39/395A61K47/48338A61K47/48423A61K47/48576A61K51/1048B82Y5/00B82Y10/00B82Y30/00C07K16/283C07K16/3007C07K16/468C12N9/96C07K2317/31C07K2317/55C07K2317/732C07K2317/734C07K2319/00C07K2319/30C07K2319/70A61K47/65A61K47/6813A61K47/6853A61P35/00A61P37/00
Inventor CHANG, CHIEN-HSINGGOLDENBERG, DAVID M.ROSSI, EDMUND A.
Owner IBC PHARMACEUTICALS INC
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